‘Silk’ has become a single, all encompassing description for an extremely wide range of biological materials (Sutherland et al., 2010) that are an ancient product in evolution. Silks are produced by a wide range insects, for example by the larvae of insects where a cocoon is formed for protection during metamorphosis, by adult insects such as webspinners that spin silk with structures on their front legs to make a web-like pouch or gallery in which they live, in the Hymenoptera (which includes bees, wasps and ants) where silk is part of nest construction, and by large numbers of other arthropods, most notably the various arachnids such as spiders where orb-webs used for catching prey are common (Sutherland et al., 2010).
Silks are fibrous protein secretions that exhibit exceptional strength and toughness and as such have been the target of extensive study. Silks are produced by over 30,000 species of spiders and by many insects. Furthermore, in comparison with other arthropods, spiders produce more than one silk type, typically between 5 and 7, each with different properties for different purposes, with most involved with web construction. Of these the major ampullate silk, also known as the ‘dragline silk’ which is used as a lifeline and for the web's outer rim and spokes has attracted much attention since it can be as strong per unit weight as steel, but much tougher.
Despite the diversity of structures and distributions, silks have features in common, notably, being semicrystalline materials, that is materials with regions of ordered molecular structure (crystallites) within an amorphous matrix and also, all show typically similar protein compositions, often rich in alanine, serine, and/or glycine (Sutherland et al., 2010).
Overall, very few of these silks have been characterised, with most research concentrating on the cocoon silk of the domesticated silkworm, Bombyx mori and on the dragline silks of the orb-weaving spider Nephila clavipes, the European garden spider, Araneus diadematus, and the nursery web spider, Euprosthenops australis. 
In the Lepidoptera and spider, the fibroin silk genes code for proteins that are generally large with prominent hydrophilic terminal domains at either end spanning an extensive region of alternating hydrophobic and hydrophilic blocks (Bini et al., 2004). Generally these proteins comprise different combinations of crystalline arrays of β-pleated sheets loosely associated with β-sheets, β-spirals, α-helices and amorphous regions (see Craig and Riekel, 2002 for review).
The ready commercial availability of domesticated silk worm, B. mori, silk, has meant that there is no commercial driver to make a recombinant silkworm silk, especially considering the difficulties in making a recombinant product for a protein that is so large and is built around a highly repetitive structure. This silk comprises 2 chains; a heavy chain of ˜390 kDa and a light chain of ˜26 kDa in a 1:1 ratio, with these 2 chains linked by a critical disulphide bond.
Very limited complete sequence data is available for spider silks. This is because of the difficulties in studying highly repetitive structures (Arcidiacono et al., 1998). The main examples include dragline silk from black widow spider (Ayoub et al., 2007) and flagelliform silk from Nephila clavipes (Hayashi and Lewis, 2000). Whereas cultivation has proved highly successful for cocoon silks, farming is not an option for spider silks. The main problem is that most spiders are very territorial, aggressive and are cannibals. This has meant, however, that these have been considerable efforts to use recombinant technologies to produce spider silks. Native spider dragline silk is remarkably strong, although dragline silks from different species show different properties, examples exist of silks that are five times stronger by weight than steel, and/or three times tougher than Kevlar (Dupont) (Gosline et al., 1999, Volrath and Knight, 2001). All dragline silks have a high MW, 250-320 kDa (Ayoub et al., 2007), which on its own provides difficulties for recombinant expression. Dragline silks are typically composed of two main proteins, the major ampulate spidroins.
Spidroins have highly repetitive structures; they are modular, and contain hundreds of tandem repeats of distinct consensus motifs. MaSp1 spidroins generally comprise two motifs, polyalanine, and GlyGlyXaa, where Xaa is frequently Leu, Tyr, Gln or Ala. MaSp2 spidroins also contain polyalanine, as well as GlyProGlyXaaXaa repeats, where Xaa is frequently Gly, Gln or Tyr. The polyalanine or poly(glycyl-alanine) sequences form into tightly packed β-sheet crystallites.
More recently, a novel structural silk from the honey bee, Apis melifera, has attracted attention. Early X-ray evidence (Rudall, 1962) and different amino acid composition suggested that distinct class of silk molecule with an alpha helical structure was present in honeybee silk. Further analysis suggested that a four-stranded coiled-coil was present (Atkins, 1967). Recent molecular studies have confirmed this (Sutherland et al., 2006). Four silk genes have been identified (AmelFibroin 1-4), that each comprising a single exon with the genes separated by short regions of 1659-1796 bases are clustered sequentially in the A. melifera genome. They do not contain the highly repetitive sequences of other silks. Hence the chains are of a size and structure that can be more readily produced by recombinant methods. Honeybee silk may be useful as a new biomedical material. For example, it can be electrospun into sheets with uniform fibres of around 200 nm.
As silk fibres represent some of the strongest natural fibres known, they have been a subject to extensive research in attempts to reproduce their synthesis. However, a recurrent problem with expression of Lepidopteran and spider fibroin genes has been low expression rates in various recombinant expression systems due to the combination of repeating nucleotide motifs that lead to deleterious recombination events, large gene size and the small number of codons for each amino acid which leads to depletion of tRNA pools. Recombinant expression leads to difficulties during translation such as translational pauses as a result of codon preferences and codon demands and extensive recombination rates leading to truncation of the genes. Shorter, less repetitive sequences would avoid many of the problems associated with silk gene expression to date.
Silks have long found applications as biomedical materials, as they are typically biocompatible, biodegradable and have low immunogenicity. For biomedical applications, recombinant silk can be fabricated into various formats. Included in these is fabrication of a natural fibre, as well as hydrogels formed using connectivity through either physical or chemical crosslinking. Variations in properties can also be produced to match specific clinical needs, so that silks that can be produced for application that need high stress prior to failure, or where extensibility is required, such as in blood vessels, and where an appropriate modulus is required to modify or control cell response, for example for tissue engineering (Vepari and Kaplan, 2007). For example, this silk has been used to form non-woven mats (Dal Pra et al., 2004), and has been electrospun into fibres and fibre mats with fibre sizes from nanometers to microns (Jin et al., 2002). Silk fibroin films can be cast from aqueous and non-aqueous solvents (Minoura et al., 1990). Porous sponges can be made from silk solutions, for example, by using salt or sugar as porogens with fibroin in HFIP (Nazarov et al., 2004) or in fully aqueous system (Kim et al., 2005).
In the early 1960s, the silk of some sawflies (Hymenopteran) was suggested to have a collagen structure by X-ray diffraction patterns obtained cocoons or from silk fibres drawn from the salivary gland of Nematus ribesii (Rudall 1967 and 1968; Lucas and Rudall, 1968). The X-ray diffraction patterns suggest twisted cables of collagen molecules with dimensions of 30 A diameter—suggesting a two or three stranded cable (Rudall, 1967 and 1968). Proteins with X-ray diffraction patterns characteristic of collagen were also found in the silk gland of other species within the tribe Nematini: in Hemichroa, Pristiphora, Pachynemalus, Pikonema and Nematus species (subfamily Nematinae); in the anterior half of the silk gland of Tomostethus and Tethida species (subfamily Blennocampinae) (Rudall and Kenchington, 1971).
After the contents of the silk gland of Nematus ribesii were dissolved in 0.2M borate buffer and then precipitated in ethanol, the following amino acid analysis (from 4 different preps) was obtained (Gly: 336 (std dev=16); Ala: 122 (2); Ser: 33 (12); Pro: 100 (3); Hydroxy-Lys: 37 (3); Lys: 14 (4): Rudall and Kenchington, 1971). This analysis indicated that the silk had a high Gly content, characteristic of collagens, along with a high Ala content. However, these amino acids are also found in high abundance in cocoon and spider silks.
Considering the unique properties of silks produced by insects, and that they are available naturally in only minute amounts, there is a need for the identification of further novel nucleic acids encoding silk proteins.